Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

Abstract

Background: Chimeric antigen receptor T (CAR-T) cell therapy, as an emerging anti-tumor treatment, has garnered extensive attention in the study of targeted therapy of multiple tumor-associated antigens in hepatocellular carcinoma (HCC). However, the suppressive microenvironment and individual heterogeneity results in downregulation of these antigens in certain patients' cancer cells. Therefore, optimizing CAR-T cell therapy for HCC is imperative.

Methods: In this study, we administered FGFR4-ferritin (FGFR4-HPF) nanoparticles to the alpaca and constructed a phage library of nanobodies (Nbs) derived from alpaca, following which we screened for Nbs targeting FGFR4. Then, we conducted the functional validation of Nbs. Furthermore, we developed Nb-derived CAR-T cells and evaluated their anti-tumor ability against HCC through in vitro and in vivo validation.

Results: Our findings demonstrated that we successfully obtained high specificity and high affinity Nbs targeting FGFR4 after screening. And the specificity of Nbs targeting FGFR4 was markedly superior to their binding to other members of the FGFR family proteins. Furthermore, the Nb-derived CAR-T cells, targeting FGFR4, exhibited significantly enhanced anti-tumor efficacy in both experiments when in vitro and in vivo.

Conclusions: In summary, the results of this study suggest that the CAR-T cells derived from high specificity and high affinity Nbs, targeting FGFR4, exhibited significantly enhanced anti-tumor efficacy in vitro and in vivo. This is an exploration of FGFR4 in the field of Nb-derived CAR-T cell therapy for HCC, holding promise for enhancing safety and effectiveness in the clinical treatment of HCC in the future.

Keywords: HCC; Nanobody; Nanoparticle; Nb-derived CAR-T cell therapy; Phage display.

Fig. 1

Construction of FGFR4 nanoparticles and immunization of alpaca. a Schematic diagram of expression vector encoding FGFR4 antigen’s extracellular domain proteins and ferritin. b Schematic diagram of generation of FGFR4-HPF nanoparticles. c Process of immunizing alpaca with FGFR4-HPF nanoparticles. d Immunized alpaca serum ELISA

Fig. 2

Construction of electroporation bacteria library and screening of anti-FGFR4 Nbs. a Monoclonal sequencing analysis and genetic evolutionary tree analysis of the electroporation library. Sequencing of 72 monoclonal clones identified 69 positive clones with insertion of single Nb sequences. b Proportion of Nb-displaying phages capable of binding to FGFR4 after screening. Left: output of first round; middle: input of second round; right: output of second round. c Monoclonal phage ELISA with FGFR4. Positive clones: 1, 9, 13, 14, 19. 20, 22, 23. d Identification of monoclonal phages capable of binding to Huh7 cells by flow cytometry. The NC phages uncapable of binding Huh7 cells were used as the control. Clones with positive fluorescence: 1 (anti-FGFR4 Nb1), 14 (anti-FGFR4 Nb2)

Fig. 3

Functional validation of the screened Nbs in vitro. a Molecular docking models of Nbs with FGFR4. The structure of light blue represents antigen, the structure of dark blue represents Nbs, the interface regions of red and green represent the region of the Nb in contact with antigen, ΔG denotes the free energy of binding. b Schematic diagram of expression vector encoding Nb-Fc. c Detection of the binding ability of Nbs by antibody-antigen binding ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by the Student’s t-test. d Detection of the binding ability of Nbs by antibody gradient dilution ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by two-way ANOVA. e Detection of the binding affinity of Nbs by SPR assay. f Evaluation the specificity of Nbs by antibody-antigen binding ELISA. Data were analyzed by one-way ANOVA. The experiments were performed independently in triplicate. Data are expressed as mean ± SEM. ****p < 0.0001

Fig. 4

Construction of Nb-derived CAR-T cells and functional validation of Nb-derived CAR-T cells anti-tumor in vitro. a Schematic diagram of lentivirus plasmid encoding anti-FGFR4 Nb-CAR. b Validation of cytotoxicity of Nb-derived CAR-T cells against Huh7 cells in vitro by LDH assay. NC-T: T cells transduced with empty lentivirus vector. c Validation of cytotoxicity of Nb-derived CAR-T cells against BXPC3 cells in vitro by LDH assay. NC-T: T cells transduced with empty lentivirus vector. d Validation of the cytokine secretion functions of Nb-derived CAR-T cells against Huh7 cells in vitro by ELISA. Mock: T cells transduced with empty lentivirus vector. e Validation of the IFN-γ secretion functions of Nb-derived CAR-T cells against Huh7 cells in vitro by ELISPOT. Mock: T cells transduced with empty lentivirus vector. Data were analyzed by two-way ANOVA. The experiments were performed independent biological replicates (N = 3). Data are expressed as mean ± SEM. Ns: p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Functional validation of the Nb-derived CAR-T cells anti-tumor in vivo. a Schematic diagram of in vivo experiments on tumor-bearing mice. b Dynamic growth curve of tumor volume (n = 4). Data were analyzed by two-way ANOVA. NC-T: T cells transduced with empty lentivirus vector. c Dynamic change curve of mouse weight (n = 4). d Validation of the proportion of the tumor-infiltrating T cells by flow cytometry. Data were analyzed by one-way ANOVA. e IHC of the tumor tissue sections stained with anti-human IFN-γ (left) and granzyme B (right) antibody. Scale bar, 100 µm. f IF of the tumor tissue sections stained with anti-human IFN-γ (green) and anti-human granzyme B (purple) antibodies, and the nuclei were stained with DAPI (blue). Scale bar, 100 µm. g H&E staining of major organs. Scale bar, 100 µm. h Kaplan Meier survival analysis of mice (n = 4). Data were analyzed by log-rank Mantel-Cox test. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01

Fig. 5

Functional validation of the Nb-derived CAR-T cells anti-tumor in vivo. a Schematic diagram of in vivo experiments on tumor-bearing mice. b Dynamic growth curve of tumor volume (n = 4). Data were analyzed by two-way ANOVA. NC-T: T cells transduced with empty lentivirus vector. c Dynamic change curve of mouse weight (n = 4). d Validation of the proportion of the tumor-infiltrating T cells by flow cytometry. Data were analyzed by one-way ANOVA. e IHC of the tumor tissue sections stained with anti-human IFN-γ (left) and granzyme B (right) antibody. Scale bar, 100 µm. f IF of the tumor tissue sections stained with anti-human IFN-γ (green) and anti-human granzyme B (purple) antibodies, and the nuclei were stained with DAPI (blue). Scale bar, 100 µm. g H&E staining of major organs. Scale bar, 100 µm. h Kaplan Meier survival analysis of mice (n = 4). Data were analyzed by log-rank Mantel-Cox test. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01